1. Field of the Invention
This invention lies in the field of assays for glycated hemoglobin.
2. Description of the Prior Art
For individuals suffering from type 1 or type 2 diabetes mellitus, maintenance of glycemic control is of prime importance, and such maintenance requires the determination of the level of hemoglobin A1c in the blood of these individuals. With diabetes reaching global epidemic proportions, it is particularly important to have accurate and reproducible HbA1c assays. HbA1c assays are also used in the screening of individuals for diabetes.
HbA1c measurements for both patient monitoring and screening are taken as an average over the lifetime of an erythrocyte. This average is compromised by several physiological conditions, notable among which are the presence of hemoglobin variants and thalassemias in the patient's blood. Hemoglobin variants are prevalent among certain ethnic groups and in certain geographical regions. Of the over 800 variants known worldwide, the most common are HbS, HbC, HbD, and HbE. HbS is most prevalent among individuals of African descent, HbD among individuals of Punjabi Indian descent, and HbE among individuals of Southeast Asia. Other known forms of hemoglobin are HbF (fetal hemoglobin) and HbA2, both of which can be elevated in thalassemia, a relatively common condition characterized by an imbalance of hemoglobin alpha and beta subunits. Beta thalassemias can also occur in the presence of HbE and HbS, and the combined sickle/beta thalassemia trait occurs most frequently among individuals of Mediterranean descent. Variants and thalassemias can cause inaccuracies in HbA1c measurements by affecting such factors as red blood cell survival and glycosylation rates. Variants also affect immunologically determined levels of glycated hemoglobin since immunoreactivity differs from one glycated variant to the next and also between glycated variants and HbA itself. Health care professionals must therefore know of the presence of variants and their proportions relative to HbA as well as the presence of thalassemias to achieve a proper determination of glycemic control.
Determinations of hemoglobin variants are typically done separately from determinations of HbA1c regardless of whether a variant is actually known to be present. Antibodies to specific variants have been developed for this purpose, and the following is a sampling of reports on such antibodies:                HbS: Jensen, R. H., et al., “Monoclonal antibodies specific for sickle cell hemoglobin,” Hemoglobin 9(4), 349-362 (1985)        HbS: Epstein, N., et al., “Monoclonal antibody-based methods for quantitation of hemoglobins: application to evaluating patients with sickle cell anemia treated with hydroxyurea,” Eur. J. Haemotol. 57(1), 17-24 (1996)        HbA: Rosenthal, M. A., et al., “Binding specificity of a monoclonal antibody to human HbA,” Hemoglobin 19(3-4), 191-196 (1995)        HbS and HbC: Garver, E. A., et al., “Screening for hemoglobins S and C in newborn and adult blood with a monoclonal antibody in an ELISA procedure,” Annals of Hematology 60(6), 334-338 (1990)        Hb with single amino acid substitutions: Stanker, L. H., et al., “Monoclonal antibodies recognizing single amino acid substitutions in hemoglobin,” J. Immunol. 136 (11), 4174-4180 (1986)        Hb variants: Moscoso. H., et al., “Enzyme immunoassay for the identification of hemoglobin variants,” Hemoglobin 14(4), 389-98 (1990)        Hb variants: Schultz, J. C., “Utilization of monoclonal antibody-based assay HemoCard in screening for and differentiating between genotypes of sickle cell disease and other hemoglobinopathies,” J. Clin. Lab. Anal. 9(6), 366-374 (1995)        
Despite these reports and others, determinations of variants are presently performed by either high performance liquid chromatography (HPLC) or electrophoresis. HPLC can indeed be a rapid means of obtaining the HbA1c level, but extended HPLC gradients are needed for detecting and quantifying the variants and thalassemias, since in HPLC impurities co-elute with the variants, and different variants tend to co-elute with each other. In fact, certain variants cannot be resolved by HPLC, even with the most optimized HPLC gradients. Typically, separate HPLC methods for rapid A1c measurements and variant and thalassemia testing are used, therefore making it impossible to simultaneously determine the A1c level and variant or thalassemia status by HPLC, much less in a rapid manner.
Assays that provide simultaneous detection of multiple analytes are termed “multiplex” assays, and disclosures of multiplex assays using affinity-type binding reactions on the surfaces of beads that are then detected by flow cytometry are disclosed in the following patents:                Watkins, M. I., et al., “Magnetic particles as solid phase for multiplex flow assays,” U.S. Pat. No. 6,280,618 B2, issued Aug. 28, 2001        Watkins, M. I., et al., “Magnetic particles as solid phase for multiplex flow assays,” U.S. Pat. No. 6,872,578 B2, issued Mar. 29, 2005        Thomas, N., “Multiple assay method,” U.S. Pat. No. 6,913,935 B1, issued Jul. 5, 2005        Hechinger, M., “Platelet immunoglobulin bead suspension and flow cytometry,” U.S. Pat. No. 6,933,106 B1, issued Aug. 23, 2005        Hechinger, M., “Anti-platelet immunoglobulin bead positive control,” U.S. Pat. No. 6,951,716 B1, issued Oct. 4, 2005        Watkins, M. I., et al., “Multi-analyte diagnostic test for thyroid disorders,” U.S. Pat. No. 7,271,009 B1, issued Sep. 8, 2007        Bell, M. L., “Assay procedures and apparatus,” U.S. Pat. No. 7,326,573 B2, issued Feb. 5, 2008        Song, Y., et al., “Multiplex protein interaction determinations using glutathione-GST binding,” US 2002/0115116 A1, published Aug. 22, 2002        
The success of multiplex assays for certain combinations of analytes does not however provide assurance, or even a high level of expectation, that similar success will be achieved for all combinations of analytes, particularly combinations with a high level of homology among the analytes. Hemoglobin and its variants and glycated forms are one such combination. Multiplex assays involve a plurality of different immunoreactants in intimate mixture in a common reaction medium, which creates competition among the immunoreactants for the different analytes, more so than in media where a single immunoreactant is present, and the cross-reactivities occur in multiple directions. The bead sets themselves must also be differentiated at the same time as the immunoassays are performed. This differentiation, whether by the use of different dyes on different bead sets, a different size for each bead set, or other known differentiation factors, adds a further level of complexity and further opportunities for cross-talk.